Intracellular calcium ion (Ca2+) plays an important role in many biological phenomena such as release of neurotransmitters in synapses, activation of ion channels in cell membrane, control of cytoplasmic enzymes, contraction of muscles (skelet al. muscle, smooth muscle, cardiac muscle), activation of leukocytes, activation of platelets and the like. They are mainly induced by a transient rise of cytoplasmic Ca2+ concentration. Therefore, an accurate measurement of intracellular Ca2+ concentration, which is free of influence on the cell functions, is important for the understanding of many life phenomena.
The cytoplasmic concentration of free Ca2+ has been measured by loading a chemically synthesized Ca2+ chelator such as Fura-2 (non-patent reference 1: Grynkiewicz, G. et al., J. Biol. Chem., 260, 3440-3450, 1985). While Fura-2 is superior in sensitivity and time responsiveness to calcium ion, it has a problem in that intracellularly introduced Fura-2 gradually leaks out from the cell with the lapse of time, and the Ca2+ sensitivity decreases with time. Since Fura-2 has a calcium ion binding activity, when the intracellular concentration of Fura-2 is raised to enhance the Ca2+ sensitivity, the dynamics of intracellular calcium ion changes.
In recent years, many Ca2+ probes using fluorescence resonance energy transfer (FRET) have been developed based on fluorescent protein by genetic engineering. There have been developed Ca2+ probes using the FRET technique, such as Cameleon (non-patent reference 2: Miyawaki, A. et al., Nature, 388, 882-887, 1997/non-patent reference 3: Miyawaki, A. et al., Proc. Natl. Acad. Sci. U.S.A., 96, 2135-2140, 1999) and FIP-CBSM (non-patent reference 4: Romoser, V. A. et al., J. Biol. Chem., 272, 13270-13274, 1997/non-patent reference 5: Persechini, A. et al., Cell Calcium, 22, 209-216, 1997), Camgaroo (non-patent reference 6: Baird, G. S. et al., Proc. Natl. Acad. Sci. U.S.A., 96, 11241-11246, 1996/non-patent reference 7: Griesbeck, O. et al., J. Biol. Chem., 276, 29188-29194, 2001), G-CaMP (non-patent reference 8: Nakai, J. et al., Nat. Biotechnol., 19, 137-141, 2001), and Pericam (non-patent reference 9: Nagai, T. et al., Proc. Natl. Acad. Sci. U.S.A. 98, 3197-3202, 2001).
Non-patent reference 2 discloses a calcium ion indicator protein obtained by the FRET technique using a combination of fluorescent proteins ECFP and EYFP, or a combination of EBFP and EGFP. A sequence of calmodulin and myosin light chain kinase has been inserted between the two fluorescent proteins. This calmodulin sequence has a calcium ion binding site in the inside, it is highly likely that the sequence influences the movement of intracellular calcium ion, and shows physiological activities such as protein modification and the like by acting on other proteins. There have been reported proteins named YC2, YC3, YC4, split YC2 and the like. When YC2, YC3 and YC4 therefrom are expressed in Hela cells, the level of response to stimulation (emission ratio: peak value of reaction/initial value) is as small as about 1.5. Split YC2 is a mixture of proteins having a shape of YC2 protein divided into two, and shows a response level of about 1.8. The longest measurement time reported in the reference is 133 min.
Non-patent reference 3 discloses a calcium ion indicator protein free of the problem of pH sensitivity in the protein described in non-patent reference 2. The basic structure of the protein is the same as that in non-patent reference 2. Therefore, this protein, too, has a calcium ion binding site in the inside, it is highly likely that the sequence influences the movement of intracellular calcium ion, and shows physiological activities such as protein modification and the like by acting on other proteins. The level of response to stimulation (emission ratio) is not improved and is about 1.5. The longest measurement time reported in the reference is 100 min.
Non-patent reference 10 (Truong, K. et al., Nat. Struct. Biol., 8, 1069-1073, 2001) discloses a calcium ion indicator protein, having improved level of response of the protein described in non-patent reference 2. The structural modification is insertion of a sequence of a calmodulin dependent kinase between calmodulin sequences. However, this protein still has a calcium ion binding site in the inside, influences the movement of intracellular calcium ion, and highly likely shows physiological activities such as protein modification and the like by acting on other proteins. The level of response is about 2.0 when a very strong stimulation of 10 μM histamine stimulation is given. This protein is not sharp in the response to stimulation, and cannot be said to accurately reflect changes in the intracellular calcium ion concentration. The longest measurement time reported in the reference is 67 min.
Non-patent reference 5 discloses a calcium ion indicator protein obtained by FRET technique using a combination of BGFP and RGFP. This protein, too, has a calcium ion binding site in the inside, it is highly likely that the sequence influences the movement of intracellular calcium ion, and shows physiological activities such as protein modification and the like by acting on other proteins. This protein shows a very weak response to stimulation.
Non-patent reference 6 discloses a calcium ion indicator fluorescent protein having a sequence wherein the former part of the EYFP amino acid sequence has been exchanged with the latter part thereof. The former part of the fluorescent protein is connected with the latter part via a calmodulin sequence. Since this protein has a calcium ion binding site in the inside, it is highly likely that the protein influences the movement of intracellular calcium ion, and shows physiological activities such as protein modification and the like by acting on other proteins. When this protein is expressed in Hela cells, the level of response upon stimulation with 200 μM histamine is about 1.5. The longest measurement time reported in the references is 13 min.
Non-patent reference 8 discloses a calcium ion indicator protein that utilizes three-dimensional structural changes of a single fluorescent protein of GFP. In this protein, a calmodulin sequence, which is also a calcium binding site, is connected to the C-terminal of a sequence wherein the former part of the amino acid sequence of EGFP is exchanged with the latter part thereof. Therefore, it is highly likely that the protein influences the movement of intracellular calcium ion, and shows physiological activities such as protein modification and the like by acting on other proteins. When this protein is expressed in HEK-293 cells, the level of response upon stimulation with 100 μM ATP is about 1.5. The longest measurement time that reported in the references is 30 min.
Non-patent reference 9 discloses a calcium ion indicator fluorescent protein having a sequence wherein the former part of the amino acid sequence of EYFP is exchanged with the latter part. The former part of the fluorescent protein is connected with the latter part via a calmodulin sequence, which is a calcium binding site. Therefore, it is highly likely that the protein influences the movement of intracellular calcium ion, and shows physiological activities such as protein modification and the like by acting on other proteins. When this protein is expressed in Hela cells, the level of response upon stimulation with 1 μM histamine is about 2.7. The longest measurement time that reported in the reference is 83 min.
On the other hand, Vanderklish et al. reported an experiment using the FRET method for visually showing active synapses. They designed a fusion protein of ECFP and EYFP using a calpain sensitive sequence as a linker, and Shaker PDZ domain sequence at the C-terminal to target the protein to postsynaptic domain (non-patent reference 11: Vanderklish, P. W. et al., Proc. Natl. Acad. Sci. U.S.A., 97, 2253-2258, 2000). Calpain is a Ca2+-activated protease found in extremely various mammalian cells (non-patent reference 12: Croall, D. E. et al., Physiol. Rev., 71, 813-847, 1991). This fusion protein is cleaved by calpain in a Ca2+ sensitive manner, and permanently loses an FRET effect when the linker peptide is cleaved. Therefore, use of the fusion protein enables identification, based on the loss of an FRET effect, of the cell having or having had an increased Ca2+ concentration due to stimulation. However, since cleavage of the linker peptide by calpain is an irreversible reaction, which makes continuous monitoring of intracellular Ca2+ concentration change unattainable, this fusion protein cannot be used as a calcium ion indicator.